Toxicity Evaluation following Intratracheal Instillation of Iron Oxide in a Silica Matrix in Rats

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Iron Oxide in a Silica Matrix in Rats

Alina Mihaela Prodan, Carmen Steluta Ciobanu, Cristina Popa, Simona

Liliana Iconaru, Daniela Predoi

To cite this version:

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Research Article

Toxicity Evaluation following Intratracheal Instillation of

Iron Oxide in a Silica Matrix in Rats

Alina Mihaela Prodan,

1

Carmen Steluta Ciobanu,

2

Cristina Liana Popa,

2,3

Simona Liliana Iconaru,

2,3,4

and Daniela Predoi

1

1_{Emergency Hospital Floreasca, Bucharest 5, 8 Calea Floreasca, Sector 1, 014461 Bucharest, Romania} 2_{National Institute of Materials Physics, 105 Bis Atomistilor, 077125 Magurele, Romania}

3_{Faculty of Physics, University of Bucharest, 405 Atomistilor, 077125 Magurele, Romania}

4_{ISTO, UMR 7327 CNRS, Université d’Orléans, 1A rue de la Férollerie, 45071 Orléans Cedex 2, France}

Correspondence should be addressed to Daniela Predoi; dpredoi@gmail.com Received 27 February 2014; Accepted 22 April 2014; Published 14 May 2014 Academic Editor: Amitava Mukherjee

Copyright © 2014 Alina Mihaela Prodan et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Iron oxide-silica nanoparticles (IOSi-NPs) were prepared from a mixture of ferrous chloride tetrahydrate and ferric chloride hexahydrate dropped into a silica xerogel composite. he structure and morphology of the synthesized maghemite nanoparticles into the silica xerogel were analysed by X-ray difraction measurements, scanning electron microscopy equipped with an energy dispersive X-ray spectrometer, and transmission electron microscopy. he results of the EDAX analysis indicated that the embedded particles were iron oxide nanoparticles. he particle size of IOSi-NPs calculated from the XRD analysis was estimated at around 12.5 nm. he average size deduced from the particle size distribution is 13.7± 0.6 nm, which is in good agreement with XRD analysis. he biocompatibility of IOSi-NPs was assessed by cell viability and cytoskeleton analysis. Histopathology analysis was performed ater 24 hours and 7 days, respectively, from the intratracheal instillation of a solution containing 0.5, 2.5, or 5 mg/kg IOSi-NPs. he pathological micrographs of lungs derived from rats collected ater the intratracheal instillation with a solution containing 0.5 mg/kg and 2.5 mg/kg IOSi-NPs show that the lung has preserved the architecture of the control specimen with no signiicant diferences. However, even at concentrations of 5 mg/kg, the efect of IOSi-NPS on the lungs was markedly reduced at 7 days posttreatment.

1. Introduction

In recent years, an increasing interest has been registered for developing the ield of nanotechnology. Due to the outstand-ing physicochemical properties that nanoparticles exhibit, the number of applications involving these nanomaterials is increasing continuously. Nowadays, nanoparticles can be

found in toothpastes, sunscreens, or food products [1,2]. he

unique chemical, physical, optical [3,4], electronic [3,5], and

magnetic [3,6] properties show that nanoparticles could be

used in biotechnology and biomedicine. he size of these nanoparticles facilitates their use in engineering of surfaces

and in creating functional nanostructures [3]. Among the

many types of nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) have been already used for several in vivo applications, showing promising results. hey were used

as contrast enhancement in magnetic resonance imaging [7–

9], for tissue repair [7, 10,11], for drug delivery in tumour

therapy [7, 12,13], for stem cell tracking [7, 14], or as heat

mediators in hyperthermia treatments [15]. In order to be

used for these types of applications, the nanoparticles bio-compatibility could be increased by adding a silica shell. Due to the biocompatible properties, silica is less likely to degrade

in a biological environment [16,17].

Cancer is a major health problem worldwide, being res-ponsible for one in four deaths in the United States,

accord-ing to the American Cancer Society [18]. In this context,

researchers have tried to ind new innovative ways of admin-istering the treatment more eiciently. One of the major pro-blems that arise during the targeted administration of drugs, most commonly used for cancer treatments, is the

nonspeci-icity of the drug towards the pathological site [3]. hus, a

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large dose of medicine is required in order for the treatment to be efective and the required local concentration to be achieved. he result consisted of the fact that besides the dam-aged cells, surrounding healthy cells could be also afected. herefore, to overcome this impediment, researchers have focused their attention on developing techniques of magnetic targeting using superparamagnetic nanoparticles. hey could be used as drug carriers, being guided by an external

mag-netic ield, thus insuring an efective treatment [3,19]. Wang

et al. showed that the iron oxide nanoparticles coated with silica can be targeted to a speciic objective via the antibody-antigen recognition by conjugation of antibodies to the outer

silica surface [20]. Coating the iron oxide nanoparticles with

various polymers such as silica, dextran, PEG, and HSA can prevent their uptake by organs. According to previous studies

[21], the silica layer on the surface of iron oxide nanoparticles

increases biocompatibility and stability against chemical degradation and can thus be used in applications such as bioseparation, genomic DNA isolation, or to control the drug release for several hours to days or months.

Another area that has attracted the interest of researchers and doctors alike is the ield of stem cell treatment. Many eforts are made in order to personalize treatments by admin-istering stem cells or genetically modiied cells. herefore, it is of great importance to trace the transplanted or injected cells and to assess their engrating eiciency and functional ability. For this purpose, SPIONs are being considered as potential

candidates [3,22].

Although the biocompatibility of iron oxide nanoparti-cles has been demonstrated, previous studies showing that once the nanoparticles come in contact with a biological medium, their surface becomes covered with diferent types

of proteins [7,23], some SPIONs with core sizes between 3

and 6 nm, coated with dextran, being approved for MRI in

patients [3, 24, 25], their biological activity can increase,

causing potential toxic interactions [26]. It has been

estab-lished that exposure to nanoparticles by people working in

automobile, aerospace, or paint industries [27–32] can lead to

major health problems, including neurotoxicity [7]. In this

context, further studies are needed in order to establish the efects on the human body by inhaled nanoparticles.

he goal of this study was to prepare iron oxide-silica nanoparticles from a mixture of ferrous chloride tetrahydrate and ferric chloride hexahydrate dropped into a silica xerogel composite. he structure and morphology of the synthesized maghemite nanoparticles into the silica xerogel were analysed by X-ray difraction (XRD) measurements, scanning electron microscopy (SEM) equipped with an energy dispersive X-ray (EDAX) spectrometer, and transmission electron microscopy (TEM). he biocompatibility of iron oxide nanoparticles embedded in a silica matrix was assessed by cell viability and cytoskeleton analysis. he biocompatibility of the iron oxide nanoparticles was evaluated using in vitro and in vivo assays, consisting in the quantiication of HepG2 cells viability. On the other hand, histological evaluation of the efect caused by the obtained nanoparticles on the lungs of male Brown Nor-way rats ater a single intratracheal instillation of a solution containing various concentrations of IOSi-NPs was per-formed in order to clarify the controversial toxicity of these nanoparticles.

2. Materials and Methods

2.1. Materials. Ferrous chloride tetrahydrate (FeCl₂⋅4H₂O),

ferric chloride hexahydrate (FeCl₃⋅6H₂O), chlorhidric acid

(HCl), ethanol, and tetraethylorthosilicate (TEOS) with 99.999% purity were purchased from Sigma-Aldrich and lead

nitrate [Pb(NO₃)₂] with 99.5% purity was purchased from

Merck.

2.2. Synthesis of Iron Oxide Nanoparticles in Silica Matrix.

he mixture of ferrous chloride tetrahydrate (FeCl₂⋅4H₂O) in

2 M HCl and ferric chloride hexahydrate (FeCl₃⋅6H₂O) with

the ratio Fe2+/Fe3+= 1/2 was dropped into a silica xerogel

composite under vigorous stirring for about 1 hour. he start-ing solution of silica xerogel composite was prepared by

mix-ing tetraethylorthosilicate, water, and ethanol [33]. he water

to TEOS and ethanol to TEOS mole ratios were 11.67 : 1 and 4 : 1. he gel was formed at room temperature under vigorous stirring. he formed gel based on iron oxide and silica was dried at room temperature. he inal product was ground to form a ine powder. he obtained nanocomposite powder was

then heat treated at 400∘C in an oven, for 24 hours (iron oxide

silica nanoparticles, IOSi-NPs).

2.3. Characterization. he X-ray difraction measurements were recorded using a Bruker D8 Advance difractometer,

with nickel iltered CuK_�(� = 1.5418 ˚A) radiation and a high

eiciency one-dimensional detector (Lynx Eye type) operated in integration mode. he difraction patterns were collected

in the 2� range 20∘–70∘, with a step of 0.02∘ and 34 s

measuring time per step.

Transmission electron microscopy (TEM) studies were carried out using a FEI Tecnai 12 equipped with a low dose digital camera from Gatan. he specimen for TEM imaging was prepared by ultramicrotomy in order to obtain thin sections of about 60 nm. he powder was embedded in an epoxy resin (polaron 612) before microtomy. he particle size was measured by the SZ-100 Nanoparticle Analyzer (Horiba) using dynamic light scattering (DLS). he signal obtained from the scattered light is fed into a multichannel correlator that generates a function used to determine the translational difusion coeicient of the particles analysed. he Stokes-Einstein equation is then used to calculate the particle size. Scanning electron microscopy (SEM) study was performed with a FEI Quanta Inspect F scanning electron microscope equipped with an energy dispersive X-ray attachment. he magnetic properties of the samples were measured using a superconducting quantum interference device (MPMS mag-netometer) at room temperature.

2.4. Cell Cultures and Conditions. he hFOB 1.19 osteoblasts cells line and the HepG2 cells were purchased from American Type Culture Collection (ATCC CCL-121, Rockville, MD, USA). he cells were routinely maintained in Dulbecco’s modiied Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% antibi-otic antimycantibi-otic solution (including 10,000 units penicillin,

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Sigma-Aldrich) at 37∘C in a humidiied atmosphere of 5%

CO₂. he cultured cells were loaded on iron oxide-silica

nanocomposite discs at a seeding density of 5× 104cells/cm2

in 24-well plates. Cells cultured in 24-well plates at the same seeding density were used as control.

2.5. In Vitro Cytotoxicity Assay. he viability of the cells was

determined by the tetrazolium salt test [32]. he medium

from each well was removed by aspiration, the cells were

washed with 200�L phosphate bufer solution (PBS)/well,

and then 50�L (1 mg/mL) of

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added on each well. Ater 2 hours of incubation, the MTT solution

from each well was removed by aspiration. A volume of 50�L

isopropanol was added and the plate was shaken to dissolve the formazan crystals. he optical density at 595 nm, for each well, was then determined using a Tecan multiplate reader (Tecan GENios, Gr¨odic, Germany). he absorbance from the wells of cells cultured in the absence of ceramic discs was used as the 100% viability value.

2.6. Analysis of the Actin Cytoskeleton. he HepG2 cells were cultured in Dulbecco’s Modiied Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and

100 U/100�g/mL penicillin/streptomycin [34] (purchased

from Invitrogen Co. (Carlsbad, CA)). Ater 24 hours of treat-ment, the medium was removed and the cells were ixed in

4% paraformaldehyde for 20 minutes at a temperature of 4∘C

and permeabilized with 0.5% Triton X-100 for 1 hour. he cells were washed with PBS ive times. Ater being washed, the cells were incubated for 45 min in PBS containing

FITC-phalloidin (20�g/mL, Sigma-Aldrich, St. Louis, MO). he

nuclei of cells were counterstained with DAPI-4�

-6-diamino-2-phenylindole (2�g/mL) for 3 min and rinsed with PBS.

Mounted slides were visualized and photographed using a luorescence microscope (Olympus BX51).

2.7. Animals. Male Brown Norway rats (weighing ∼300 ±

10 g) were purchased from the National Institute of Research and Development for Microbiology and Immunology “Can-tacuzino,” Bucharest. he rats were housed in an environment

controlled for temperature (22± 2∘C), light (12 h light/dark

cycles), and humidity (60± 10%). he animals were

main-tained under speciic pathogen-free conditions in accordance with NIH Guide for the Care and Use of laboratory Animals. 2.8. Intratracheal Instillation. Ater acclimatization for one

week, the rats were randomly divided into four groups (� = 4

per group) to receive vehicle control (saline, 0.9% NaCl, 0.3 mL) or instillation of a solution containing 0.5, 2.5, or 5 mg/kg IOSi-NPs. he rats were anesthetized with sodium methohexital (35 mg/kg, intraperitoneally) and placed on an inclined restraint board before instillation with saline suspension or IOSi-NPs. he IOSi-NPs were suspended in physiological saline solution. Before intratracheal instillation, the IOSi-NPs suspension was ultrasonicated for 30 min. he instillation was performed using a nonsurgical intratracheal

instillation method [35] adapted from Hatch et al. [36]. he

rats were anesthetized by ether. According to Bai et al. [35],

a ball tripped needle was maneuvered through the epiglottis, ater which contact with the tracheal rings provides conirma-tion that the needle is, in fact, within the trachea. Aterwards,

an injector with 100�L physiological saline or IOSi-NPs

suspension was inserted into the ball tripped needle. To allow the luid to drain into the respiratory tree, ater the physio-logical saline or IOSi-NPs suspension gently instilled into the trachea, the animal was maintained in an upright position for 5 min. he rats were euthanized and sacriiced ater 24 h and one week from the instillation, according to the Guide for the Care and Use of Laboratory Animals. All animals were humanely treated and were monitored for any potential sufering.

2.9. Histological Examination. Histopathology analysis was performed in Floreasca Emergency Hospital, Bucharest, Romania. he lungs derived from the rats in the control group and the treated groups were ixed with 10% neutral bufered formalin and processed using routine histological techniques.

Ater parain embedding, 3�m sections were cut and stained

with hematoxylin and eosin (H and E) for histopathologic evaluation. he morphological changes were observed under

the microscope [37].

3. Results and Discussions

he IOSi-NPs have been widely used for environmental, bio-logical, and medical applications but their potential toxicity at nanometric scale provides a growing concern about the risk factor for human health. In order to assess the risks of IOSi-NPs, the objective of this study was to evaluate and compare the pulmonary responses induced in rats ater intratracheal instillation of suspensions containing various concentrations of IOSi-NPs.

Before carrying out the study on evaluating and compar-ing pulmonary responses induced in rats ater intratracheal instillation of suspensions containing various concentrations of IOSi-NPs, characterization of the synthesized ultraine IOSi-NPs particles was performed using XRD analysis, EDAX analysis, scanning electron microscopy, and transmis-sion electron microscopy.

Good pattern it has been achieved by the Rietveld method using MAUD (material analysis using difraction)

[38]. he XRD analysis of iron oxide-silica nanocomposite

Rietveld reinement of X-ray difraction patterns revealed a phase mixture of maghemite and amorphous silica. he com-parison between the experimental and calculated data obtained with the joint Rietveld reinement of the sample is

presented inFigure 1.

he experimental data in blue and the calculated data are represented by a grey line. Vertical lines represent the posi-tions of difraction lines of maghemite and amorphous silica. he line below the grey plot is the diference proile. It resulted in that each sample is constituted of spherical

nanocrystal-lites. he difraction peak at about 2� = 23 is related to

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Int en sit y 1/ 2 (co un t 1/ 2 ) 20 40 60 115 110 105 Silica glass Maghemite 2-�(deg)

Figure 1: Experimental (blue), calculated (solid line gray), and diference plot (lover line) of�-Fe₂O₃and silica.

(511), and (440) agrees with the cubic structure of maghemite in Fd3m space group (ICSD-PDF no. 79196) with a lattice

parameter of 8.35 ˚A. he XRD showed a slight broadening

of the difraction lines which can be interpreted in terms of

small sized crystallites [39]. he calculated particle size of

maghemite silica nanocomposites was estimated at around 12.5 nm. Based on the XRD data reinement, the formation of single-phase spinel cubic structure belonging to the Fd3m space group has been conirmed. he progress of the Rietveld reinement for the sample is monitored by a number of

agreement parameterssuch as the weighted proile�wpindex

and the “goodness of it” (�) index which is the ratio of �wp

over the statistically expected�exp.

On the other hand, in the Rietveld method,�Braggis useful

because it depends on the it of the structural parameters and

not on the proile parameters. he inal � factors [40]

obtained from the analysis of the iron oxide-silica

nanocom-posites were given by�wp = 1.0058 (%) and �exp = 0.9530

(%). he resulting Bragg�-factor and chi squared (�2) were

0.58 and 1.1138, respectively. heoretical values of the �

factors obtained for maghemite-silica nanocomposites are in

good agreement with the theory of Toby [41].

Information about the size and typical shape of the

IOSi-NPs obtained ater heat treatment at 400∘C of the obtained

initial nanocomposites powder based iron oxide and silica was provided from SEM analysis. SEM image of maghemite-silica nanocomposites showed very small particle sizes and

uniform spherical shapes (Figure 2(a)). EDAX spectrum

and elemental maps (Figure 2(b)) of Fe, O, and Si for the

maghemite-silica nanocomposites are also presented. he uniform distributions of Fe, O, and Si could be observed.

he morphological investigation of iron oxide-silica nanoparticles was performed using transmission electron

microscopy (TEM).Figure 3shows the TEM image of

IOSi-NPs, the selected area electron difraction (SAED), and size distribution of the particles. Details observed at high magniication (180000x) show monodisperse IOSi-NPs with spherical shape and a monomodal particle size distribution. he average size deduced from the particle size distribution is 13.7 ± 0.6 nm, which is in good agreement with XRD analysis. Dynamic light scattering known as Quasi-Elastic Light Scattering was used to determine the size distribution proile

of small maghemite-silica particles in suspension. he mag-hemite-silica nanoparticles observed by DLS are

monodis-perse in water (Figure 4). he size of IOSi-NPs deduced from

TEM images is consistent with the DLS size proiles (14 nm). Magnetic measurements of the iron oxide-silica nanopar-ticles were carried out using vibrating sample magnetometer (VSM). Magnetization curves (M-H loop) for the IOSi-NPs

obtained ater heat treatment at 400∘C measured at room

temperature are presented inFigure 5.

According to Dormann et al. [42], iron oxide-silica

nanoparticles keep the superparamagnetic characteristic of the iron oxide nanoparticles. From hysteresis measurements, the zero coercivity ields of IOSi-NPs can be considered equal to 0 Oe at 300 K. he very little value of coercivity ields

observed inFigure 5(b)can be attributed to the fact that iron

oxide nanoparticles in silica matrix do not have the ability to

rotate freely. In agreement with Im et al. [43], the magnetic

domain sizes of iron oxide nanoparticles in silica matrix can be easily over the limit of superparamagnetism. According

to previous studies [43, 44], the silica colloids at room

temperature exhibit a very small coercivity. On the other hand, the value of saturation magnetizations, Ms, was about

22.29 emu/g. In previous studies, Pareta et al. [45] have

attributed the low saturation magnetization of iron oxide in

silica matrix to the diamagnetic contribution of the SiO₂

shells covering the iron oxide nanoparticles, thus weakening

the magnetic moment for iron oxide/SiO₂nanoparticles due

to the occurrence of thicker shells.

In order to quantitatively evaluate the hFOB 1.19

osteoblast cell viability, we performed MTT assay (Figure 6).

he cell viability of hFOB 1.19 osteoblasts cells in the presence of various concentrations of IOSi-NPs (concentrations from

25 to 100�g/mL) ater 24 h indicated that osteoblast viability

was similar in the presence of IOSi-NPs compared to the

control, as shown inFigure 6(a). Ater exposure to increasing

concentrations from 25 to 100�g/mL of IOSi-NPs for 48 h,

the cell viability of hFOB 1.19 osteoblasts cells is not

inlu-enced by the presence of IOSi-NPs (Figure 6(b)).

In the present study, ater 24 h and 48 h from exposure with IOSi-NPs, there could not be observed higher values of cell viability in the presence of IOSi-NPs (concentrations

from 25 to 100�g/mL) compared to the control. It was thus

proved that the IOSi-NPs remain dispersed in cell cul-ture, keeping bioactivity for all concentrations from 25 to

100�g/mL [45]. he cytotoxicity test by MTT assay, using

hFOB 1.19 osteoblasts cells, has indicated that the studied IOSi-NPs were nontoxic to the cells.

On the other hand, we tested the cell viability of HepG2 cells in the presence of various concentrations of IOSi-NPs and the expression of F-actin in HepG2 cells adhered on IOSi-NPs nanoparticles. Ater exposure to increasing

con-centrations from 25 to 100�g/mL of IOSi-NPs for 24 h and

48 h, the cell viability of HepG2 cells was measured using

MTT assay (Figure 7). In order to investigate the cell viability,

it is relevant to note that in all the studies, the cells are not inluenced by the presence of IOSi-NPs, preserving a good morphology adhesion. When the concentration of IOSi-Nps increased, the cell variability decreased from 100%

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(a) 2.3 6.3 (keV) Fe Si O (b)

Figure 2: SEM micrographs (a) and elemental maps of maghemite-silica nanocomposite.

(a) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 5 10 15 20 F re q uenc y (%) Diameter (nm) (b)

Figure 3: TEM micrograph (high magniication at 180000x) showing the spherical IOSi-NPs, selected area electron difraction (SAED), and size distribution. 1 10 100 1000 In te n si ty (a.u .) Size (nm)

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0 2 4 6 0 5 10 15 20 25 M agnetiza tio n (em u/g) Magnetic field (T) −6 −4 −2 −25 −20 −15 −10 −5 (a) 0 0.1 0.2 M agnetiza tio n (em u/g) Magnetic field (T) 0 2 4 6 8 10 −0.2 −0.1 −10 −8 −6 −4 −2 (b)

Figure 5: M-H curves of IOSi-NPs at room temperature in full scale (a) and in extended scale (b).

0 20 40 60 80 100 Control 25 50 75 100 Ce ll v ia b il it y ( % )

MTT 24 h, hFOB 1.19 osteoblast cells

Concentration of IOSi-NPs (𝜇g/mL) (a) 0 20 40 60 80 100 Control 25 50 75 100 C el l via b ili ty (% )

MTT 48 h, hFOB 1.19 osteoblast cells

Concentration of IOSi-NPs (𝜇g/mL)

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Figure 6: Cell viability data assessed by a MTT assay for hFOB 1.19 osteoblast cells incubated for 24 h (a) and 48 h (b) with the IOSi-NPs at various concentrations (25, 50, 75, and 100�g/mL).

variability decreased to 92% when the IOSi-NPs

concentra-tion is equal to 100�g/mL [46]. Furthermore, ater 48 hours

of exposure with IOSi-NPs, we observed a tendency of linear

increase of viability and proliferation (Figure 7).

his efect might be due to cells adaptation at interaction with IOSi-NPs nanocomposites. According to Weichsel et al.

[47], the modeling and spatial organization of the actin

cyto-skeleton is a very active and increasingly sophisticated research.

he functions of the actin cytoskeleton are to mediate a variety of essential biological functions in all eukaryotic cells, including intra- and extracellular movement and structural support. In order to perform these functions, the organiza-tion of the actin cytoskeleton must be tightly regulated both

temporally and spatially [48]. According to previous studies

on the size efect on cell uptake in well-suspended, uniform

mesoporous silica nanoparticles [49], facile synthesis of

monodispersed mesoporous silica nanoparticles with ultra

large pores and their application in gene delivery [50] and

silica-based complex nanorattles as multifunctional carrier

for anticancer drug [51] showed that for intracellular drug

delivery and eicient therapy, an eicient cellular internaliza-tion of nanoparticles is necessary.

For detection of F-actin, the major constituent of micro-ilaments (green), the cells were ixed and stained with

FITC phalloidin (Figure 8). he cells were stained with

4,6-diamidino-2-phenylindole dihydrochloride (DAPI) in order to visualize cell nuclei.

Ater cells treatment with the IOSi-NPs, there could not be found any morphological changes with observable frag-mented nuclei. Moreover, staining of F-actin and nuclear staining have shown that the assimilation of the nanoparticles did not have any efect on nuclear morphology nor on the cytoskeleton of transfected cells.

According to previous studies [52,53], at this stage, little is

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0 20 40 60 80 100 Control 25 50 75 100 C ell via b ili ty (%) MTT 24 h Concentration of IOSi-NPs (𝜇g/mL) (a) 0 20 40 60 80 100 Control 25 50 75 100 C ell via b ili ty (% ) MTT 48 h Concentration of IOSi-NPs (𝜇g/mL) (b)

Figure 7: Cell viability data assessed by a MTT assay for HepG2 cells incubated for 24 h (a) and 48 h (b) with the IOSi-NPs at various concentrations (25, 50, 75, and 100�g/mL). Merge F-actin DAPI 24 h 48 h Co n tr o l Co n tr o l 50 𝜇 g/mL 100 𝜇 g/mL 50 𝜇 g/mL 100 𝜇 g/mL

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(a) (b)

(c) (d)

Figure 9: Light optical image of the lung at 24 h ater intratracheal instillation of IOSi-NPs in rats at various concentrations. he reference sample is also presented (a). Lung ater 24 hours: control (a), 0.5 mg/kg (b), 2.5 mg/kg (c), and 5 mg/kg (d).

to these airborne nanoparticles. From this point of view, the potential toxic efects of IOSi-NPs by intratracheal instillation are discussed. Although it has been shown that the iron oxide and silica nanoparticles have an excellent potential for biomedical applications, there is not an understanding of their potential systemic toxicity. According to the previous

studies of Drobne [54] and Seaton [55], the particular

nanoscale properties are likely to afect not only the chemistry and physics but also their behaviour in biological systems. he toxicity evaluation ater 24 h and 7 days from intratra-cheal instillation of various concentrations of IOSi-NPs in

rats was performed by histopathological analysis (Figures9–

12). All animals survived the administration of IOSi-NPs on

all the tested concentrations and did not show any sign of discomfort (lethargy, nausea, vomiting, or diarrhea) during the whole duration of the experiment. he histopathological assessment of the selected tissues such as lung and liver was conducted.

he toxicity evaluation of the lung ater 24 h from intratracheal instillation of various concentrations of IOSi-NPs in rats was observed by histopathological

investiga-tions (Figure 9). Ater 24 h from intratracheal instillation,

the rats showed particle-induced modiications that were dependent on the concentrations used. Ater 24 h from the intratracheal instillation with 0.5 mg/kg of IOSi-NPs, the lung parenchyma of the rats showed preserved alveolar architec-ture with rare macrophages in the alveolar septa. We could see that the pathological micrographs of lung in rats ater the intratracheal instillation with 0.5 mg/kg dose of IOSi-NPs

(Figure 9(b)) show that the lung has preserved the

architec-ture of the control specimen (Figure 9(a)), with no signiicant

diferences. Ater the intratracheal instillation of the rats

with a 2.5 mg/kg (Figure 9(c)) dose of IOSi-NPs, the lung

parenchyma of the rats showed preserved alveolar architec-ture with rare macrophages in the alveolar septa, discreet anisokaryosis, and anisochromia of type II pneumocytes with rare nucleoli. Lung parenchyma of the specimen ater intratracheal instillation of IOSi-NPs in rats at concentration

of 5 mg/kg (Figure 9(d)) showed preserved alveolar

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(a) (b)

(c) (d)

Figure 10: Light optical image of the lung at 7 days ater intratracheal instillation of IOSi-NPs in rats at various concentrations. he reference sample is also presented (a). Lung ater 7 days: control (a), 0.5 mg/kg (b), 2.5 mg/kg (c), and 5 mg/kg (d).

However, the efect of IOSi-NPS on lungs, even for concentration of 5 mg/kg, was markedly reduced ater 7 days of treatment. Ater 7 days from intratracheal instillation of the

rats with 5 mg/kg IOSi-NPs (Figure 10(d)), we observed that

the lung parenchyma show preserved alveolar architecture with rare macrophages, discreet anisokaryosis, and anisoch-romia of type II pneumocytes, with rare chromocenters and

nucleoli (Figure 10(d)). On the other hand, the pathological

micrographs of lungs in rats ater the intratracheal instillation with 0.5 mg/kg and 2.5 mg/kg dose of IOSi-NPs (Figures

10(b)-10(c)) show that the lung has preserved the architecture

of the control specimen (Figure 10(a)) with no signiicant

diferences.

According to the literature [56], the liver is the irst

organ exposed to nanoparticles that are capable to enter into the circulation ater intratracheal instillation because it is the major organ for biotransformation of toxins. he liver examination ater intratracheal instillation of the rats with

various concentrations of IOSi-NPs (Figure 11) indicates that

the liver has preserved the architecture of the control

spec-imen (Figure 11(a)) with no signiicant diferences even for

values of 5 mg/kg.

Ater microscopic examination of the liver and spleen 24 h from intratracheal instillation of IOSi-NPs in rats, we can see that none of the organs other than the lung revealed any related toxic response, in good agreement with previous

studies [57].

Previous studies [58, 59] suggested that smaller sized

nanoparticles could initiate lung injury such as the inlam-mation in lung, which induced endothelial-epithelial damage and subsequent iniltrated leukocytes that allowed large amounts of smaller nanoparticles to pass easily into circula-tion. Moreover, in the lung injury, the size and concentrations of nanoparticles play a major role.

Iron oxide nanoparticles were developed rapidly and because of their superparamagnetic characteristics, they are of considerable interest for potential applications in biology and medicine, magnetic target drug delivering, and environ-mental catalysis. According to previous studies of Wu et al.

[60], the surface functionalized magnetic iron oxide

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(a) (b)

(c) (d)

Figure 11: Light optical image of the liver at 24 h ater intratracheal instillation of IOSi-NPs in rats at various concentrations. he reference sample is also presented (a). Liver ater 24 hours: control (a), 0.5 mg/kg (b), 2.5 mg/kg (c), and 5 mg/kg (d).

evaluate the potentially toxic efects of IOSi-NPs on the lungs, spleen, and liver of rats ater being exposed for 24 h and 7 days, respectively, to a single intratracheal instillation of solutions containing concentrations of 0.5, 2.5, or 5 mg/kg.

Consistent with other reports examining various

materi-als like CeO₂[61,62], titanium dioxide [63], silica [64], and

copper [65] nanoparticles, our data suggest that IOSi-NPs are

not capable of translocating from the lungs to the liver via the circulatory system at all the tested concentrations. he his-topathological appearance of the liver ater intratracheal instillation of IOSi-NPs in rats shows that the diferent pathological alterations such as enlargement of hepatocytes, hydropic degeneration of hepatocytes, nuclear enlargement, and dilatation of the sinusoids were not observed at concen-trations of 0.5, 2.5, and 5 mg/kg.

4. Conclusions

he purpose of our research focuses on the development of various strategies of synthesis and control of the structure and magnetic properties of surface functionalized iron oxide

nanoparticles and their corresponding applications. Iron oxide nanoparticles were encapsulated in a silica matrix. Silica coated magnetic nanoparticles have been previously synthesized by adding into a silica xerogel composite the mix-ture of ferrous chloride tetrahydrate and ferric chloride hex-ahydrate. A systematic study of the formation of iron oxide coated with silica was made. he IOSi-NPs characterized by various techniques were found to have a narrow size distribu-tion with an average size deduced from TEM measurements

of around 13.7± 0.6 nm in good agreement with the size

deduced from the XRD analysis that was estimated at around 12.5 nm.

In terms of the viability, it can be noted that in all the studies, the cell viability was not inluenced by the presence of IOSi-NPs, preserving a good morphology adhesion.

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(a) (b)

(c) (d)

Figure 12: Light optical image of the spleen at 24 h ater intratracheal instillation of IOSi-NPs in rats at various concentrations. he reference sample is also presented (a). Spleen ater 24 hours: control (a), 0.5 mg/kg (b), 2.5 mg/kg (c), and 5 mg/kg (d).

In conclusion, we demonstrated that intratracheal expo-sure with doses containing 0.5 mg/kg, 2.5 mg/kg, and 5 mg/kg IOSi-NPs did not initiate acute lung injury and that the synthesized nanoparticles with sizes around 12.5 nm are not able to enter into the circulatory system.

In this study we tried to answer some questions of the currently pressing problems regarding the toxicity of nano-particles and their behaviour in vitro and in vivo. For a better understanding of the behaviour of these nanoparticles on health more complex studies will have to be carried out in the future on toxicity induced ater intratracheal instillation of ine and ultraine particles.

Conflict of Interests

he authors declare that there is no conlict of interests regarding the publication of this paper.

Acknowledgment

his work was inancially supported by the IFA-CEA program under Project no. C206.

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